The present invention is directed to compounds which are useful as inhibitors of metalloproteases, e.g. zinc proteases, particularly zinc hydrolases, and which are effective in the prophylaxis and treatment of disease states which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage, Raynaud syndrome and pulmonary high pressure. In addition the compounds are useful as cytostatic and cerebroprotective agents for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
Endothelins are peptides, that exist in three isoforms ET-1, ET-2, and ET-3, each encoded by a distinct gene. They have been originally discovered in the conditioned medium of porcine endothelial cells in 1988 by Yanagisawa (Yanagisawa M; Kurihara H; Kimura S; Tomobe Y; Kobayashi M; Mitsui Y; Yazaki Y; Goto K; Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells [see comments]. NATURE (1988 Mar 31), 332(6163), 411-5.). The active ETs are peptides of 21 amino acids with two intramolecular disulfide bridges. They are produced from preproproteins of 203 to 212 amino acids which are processed by furin like endopeptidases to the biologically inactive big-endothelin (big-ET). The big-ETs are specifically processed to mature ETs by a hydrolytic cleavage between amino acids 21 and 22 that are Trp21-Val22 (big-ET-1, big ET-2) and Trp21-Ile22 in big-ET-3 respectively. Already in 1988 a specific metalloprotease was postulated to be responsible for this specific cleavage. In 1994 ECE-1 (endothelin converting enzyme-1) was purified and cloned from bovine adrenal (Xu D, Emoto N, Giaid A, Slaughter C, Kaw S, de Witt D, Yanagisawa M: ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell (1994) 78: 473-485.).
ECE-1 is a membrane bound type II zinc-endopeptidase with a neutral pH optimum and a zinc binding motif HExxHx( greater than 20)E. It belongs to subfamily M13 and has a large 681 amino acid ectodomain that comprises the active site. Other members of the M13 family are NEP24.11 (neutral endopeptidase), PEX, a phosphate regulating neutral endopeptidase, and Kell blood group protein that has recently been described as a big-ET-3 processing enzyme. Members of the M13 family of human origin are characterized by a high molecular weight ( greater than 80 kDa) a number of conserved disulfide bridges and a complex glycosylation pattern. The structure of NEP has recently been solved. (Oefner et al, J. Mol. Biol. 2000, 296, 341-349). The catalytic domain of ECE and related human M13 proteinases are significantly larger ( greater than 650 amino acids) than members of matrix metalloproteases (MMPs). Unlike the family of the MMPs which belong to the metzincins and display a typical HExxHxxGxxH pattern members of the M13 family are gluzincins comprising a HExx( greater than 20)E pattern. These two families are clearly different in size of catalytic domains, structure and zinc coordinating pattern of ligands. Active sites of the two families show clear differences which has clear impact on type of inhibitors and the potential selectivity.
The invention is directed to a compound of formula I 
wherein
R1 is hydrogen, alkylcarbonyl, or arylcarbonyl;
R2 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl, heterocyclyl or hetercycylalkyl;
A is xe2x80x94C(O)xe2x80x94R3, xe2x80x94CH(OH)xe2x80x94R4, or xe2x80x94C(O)xe2x80x94NR5R6, wherein
R3 and R4 are independently alkyl, aryl, arylalkinyl, arylalkyl, or arylalkenyl;
R5 is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyalkyl, or arylalkyl;
R6 is alkyl, alkylcarbonylalkyl, cyanoalkyl, hydroxyalkyl, hydroxyalkyl-(hydroxyalkyl), alkoxycarbonylalkyl, arylalkyl, arylcarbonylalkyl, arylaminocarbonylalkyl, aryl(alkyl)aminocarbonylalkyl, aminocarbonylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, cycloalkyl, cycloalkylalkyl, or carboxyalkyl, or R6 is formula IIa, IIb, or IIc; 
xe2x80x83or
xe2x80x94NR5R6 in xe2x80x94C(O)xe2x80x94NR5R6 for A represents a 5 or 6 membered saturated ring, unsubstituted or substituted with carboxy, alkyloxycarbonyl, hydroxy, alkoxycarbonylalkoxy, phenylalkyl, or phenylalkoxycarbonyl;
R7 is hydrogen, alkyl, alkenyl, alkylthioalkyl, aryl, heteroaryl, carboxyalkyl, carboxy, alkoxycarbonylalkyl, arylalkyl, or heteroarylalky; or a compound of formula III 
R7A is hydrogen or alkyl;
R8 is xe2x80x94OR9 or xe2x80x94NR10R11, wherein
R9 is hydrogen, alkyl, arylalkyl; R10 is hydrogen or alkyl; and R11 is alkyl, aryl, heteroaryl, arylalkyl, or the group xe2x80x94NR10OR11 represents a 5 or 6 membered ring unsubstituted or substituted with carboxy, alkyloxycarbonyl, hydroxy, alkoxycarbonylalkoxy, phenylalkyl, or phenylalkoxycarbonyl;
R12 is alkyl, aryl, or arylalkyl;
Y isxe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(O2)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94;
m is 0, 1, or 2;
X is xe2x80x94SO2, xe2x80x94COxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94SO2NHxe2x80x94, or xe2x80x94C(O)NR13xe2x80x94 wherein R13 is hydrogen, alkyl, aryl, or carboxyalkyl;
or a dimeric form, or a pharmaceutically acceptable ester, or a pharmaceutically acceptable salt thereof.
The term xe2x80x9calkylxe2x80x9d, alone or in combination, means a straight-chain or branched-chain alkyl group containing a maximum of 7, preferably a maximum of 4, carbon atoms, e.g., methyl, ethyl, n-propyl, 2-methylpropyl (iso-butyl), 1-methylethyl (iso-propyl), n-butyl, and 1,1-dimethylethyl (t-butyl).
The term xe2x80x9ccarboxyxe2x80x9d refers to the group xe2x80x94C(O)OH.
The term xe2x80x9ccarbonylxe2x80x9d refers to the group xe2x80x94C(O)xe2x80x94.
The term xe2x80x9chalogenxe2x80x9d refers to the group fluoro, bromo, chloro and iodo.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic double bond (including for example, vinyl, allyl and butenyl).
The term xe2x80x9calkinylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one olefinic triple bond (including for example propinyl, butin-(1)-yl, etc.).
The term xe2x80x9calkoxyxe2x80x9d, alone or in combination, means an alkyl ether group in which the term xe2x80x98alkylxe2x80x99 has the significance given earlier, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy, tert.butoxy and the like.
The term xe2x80x9calkoxycarbonylxe2x80x9d refers to a group of the formula xe2x80x94C(O)Rc wherein Rc is alkoxy as defined above.
The term xe2x80x9chydroxyxe2x80x9d refers to the group xe2x80x94OH, the term xe2x80x9ccyanoxe2x80x9d to the group xe2x80x94CN.
The term xe2x80x9chydroxyalkylxe2x80x9d means an alkyl group as defined earlier which is substituted by a hydroxy group.
The term xe2x80x9cthioalkylxe2x80x9d and xe2x80x9ccyanoalkylxe2x80x9d refer to an alkyl group as defined earlier which is substituted by a xe2x80x94SH group or an xe2x80x94CN group, respectively.
The term xe2x80x9chalogenalkylxe2x80x9d refers to an alkyl group as defined earlier which is substituted by one to three halogen atoms, preferably fluoro, e.g. trifluoromethyl, 2,2,2-trifluoroethyl, etc.
The term xe2x80x9calkylthioalkylxe2x80x9d is a group of the formula alkyl-S-alkyl.
xe2x80x9cCarboxyalkylxe2x80x9d means a lower-alkyl as defined above which is substituted by a HOOC-group.
The term xe2x80x9calkylcarbonylxe2x80x9d, alone or in combination, means an acyl group derived from an alkanecarboxylic acid, i.e. alkylxe2x80x94C(O)xe2x80x94, such as acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl etc.
The term xe2x80x9ccycloalkylxe2x80x9d signifies a saturated, cyclic hydrocarbon group with 3-8, preferably 3-6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like.
The term xe2x80x9caminoxe2x80x9d refers to the group xe2x80x94NH2.
The term xe2x80x9carylxe2x80x9d for R2xe2x80x94alone or in combinationxe2x80x94, refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl, tetrahydronaphthyl, fluorenyl or biphenyl, preferably phenyl or naphthyl, and most preferably naphthyl. The aryl moiety, especially phenyl, is unsubstituted or substituted with one or more groups independently selected from halogen, preferably fluoro, alkyl, alkoxy, mono- or dialkylamino, carboxy, alkoxy, and alkylcarbonylamino.
The term xe2x80x9cheteroarylxe2x80x9d for R2xe2x80x94alone or in combinationxe2x80x94refers to an aromatic monoxe2x80x94or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are thiophenyl, isoxazolyl, thiazolyl, pyridinyl, pyrrolyl, imidazolyl, tetrazolyl, preferably thiophenyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with alkyl, alkylcarbonyl, halogen, preferably fluoro, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl. The preferred heteroaryl group is thiophenyl.
The term xe2x80x9carylxe2x80x9d for R1 and R3 to R12xe2x80x94alone or in combinationxe2x80x94refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl. The aryl moiety is optionally substituted with one or more groups independently selected from halogen, preferably fluoro, alkoxycarbonyl, e.g. methylcarbonyl, alkyloxycarbonylalkoxy, carboxy, carboxyalkoxy, cyano, alkyl, alkoxy, phenyl, phenoxy, phenylalkyl, phenylalkoxy, trifluormethyl, trifluormethoxy, hydroxy, alkylamido, e.g. acetamido, nitro, alkylsulfonyl, e.g. methylsulfonyl. The preferred substituents are fluoro, carboxy, alkyloxycarbonyl, hydroxy, hydroxyalkyl, alkoxycarbonylalkoxy, carboxyalkyl, and carboxyalkoxy.
The term xe2x80x9caryloxyxe2x80x9d refers to an aryl group as defined above attached to a parent structure via an oxy radical, i.e., arylxe2x80x94Oxe2x80x94.
The term xe2x80x9cheteroarylxe2x80x9d for R3 and R4 to R10xe2x80x94alone or in combinationxe2x80x94refers to an aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are thiophenyl, isoxazolyl, thiazolyl, pyridinyl, pyrrolyl, imidazolyl, tetrazolyl, indolyl, benzoimidazolyl, oxadiazolyl, preferably pyridinyl, isoxazolyl, benzodioxolyl and thiazolyl, preferably indolyl, tetrazolyl, benzoimidazolyl, oxadiazolyl, and benzodioxolyl. Optionally, the heteroaryl group can be mono-, di- or tri-substituted, independently, with halogen, alkyl, alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino, dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl or halogen, preferably fluoro.
The term xe2x80x9cheterocyclylxe2x80x9d xe2x80x94alone or in combinationxe2x80x94refers to a non-aromatic mono- or bicyclic radical having 5 to 10, preferably 5 to 6 ring atoms, containing one to three heteroatoms, preferably one heteroatom, e.g. independently selected from nitrogen, oxygen or sulfur. Optionally the heterocyclic ring can be substituted by a group independently selected from halogen, alkyl, alkoxy, oxocarboxy, alkoxycarbonyl, etc. and/or on a secondary nitrogen atom (i.e. xe2x80x94NHxe2x80x94) by alkyl, arylalkoxycarbonyl, alkylcarbonyl or on a tertiary nitrogen atom (i.e. xe2x95x90Nxe2x80x94) by oxido. Examples for heterocyclic groups are morpholinyl, pyrrolidinyl, piperidyl, etc.
The term xe2x80x9cdimeric formxe2x80x9d means a compound wherein the two R1 groups of two identical compounds of formula I have been replaced by a common single bond or wherein R1 is glutathione-Sxe2x80x94 or cysteine-Sxe2x80x94 or ester and/or alkylcarbonyl or arylcarbonyl derivatives thereof, e.g. acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94, preferably glutathione-Sxe2x80x94, cysteine-Sxe2x80x94, acetylcysteine-Sxe2x80x94 or benzoylcysteine-Sxe2x80x94.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein and the like. In addition these salts may be prepared by addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts and the like. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polymine resins and the like.
xe2x80x9cPharmaceutically acceptable estersxe2x80x9d means that compounds of general formula (I) may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compounds in vivo. Examples of such compounds include physiologically acceptable and metabolically labile ester derivatives, such as methoxymethyl esters, methylthiomethyl esters and pivaloyloxymethyl esters. Additionally, any physiologically acceptable equivalents of the compounds of general formula (I), similar to the metabolically labile esters, which are capable of producing the parent compounds of general formula (I) in vivo, are within the scope of this invention.
The compounds of formula (I) are useful in inhibiting mammalian metalloprotease activity, particularly zinc hydrolase activity. More specifically, the compounds of formula (I) are useful as medicaments for the treatment and prophylaxis of disorders which are associated with diseases caused by endothelin-converting enzyme (ECE) activity. Inhibiting of this enzyme would be useful for treating myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma. In addition the compounds are useful as cytostatic and cerebroprotective agents, for inhibition of graft rejection, for organ protection and for treatment of ophthalmological diseases.
In more detail, the present invention relates to compounds of formula (I) 
wherein
R1 is hydrogen, alkylcarbonyl, or arylcarbonyl;
R2 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonylalkyl, alkinyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, aryl(alkoxycarbonyl)alkyl, heteroaryl, heteroarylalkyl, heterocyclyl or hetercycylalkyl;
A is xe2x80x94C(O)xe2x80x94R3, xe2x80x94CH(OH)xe2x80x94R4, or xe2x80x94C(O)xe2x80x94NR5R6, wherein
R3 and R4 are independently alkyl, aryl, arylalkinyl, arylalkyl, or arylalkenyl;
R5 is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyalkyl, or arylalkyl;
R6 is alkyl, alkylcarbonylalkyl, cyanoalkyl, hydroxyalkyl, hydroxyalkyl-(hydroxyalkyl), alkoxycarbonylalkyl, arylalkyl, arylcarbonylalkyl, arylaminocarbonylalkyl, aryl(alkyl)aminocarbonylalkyl, aminocarbonylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, cycloalkyl, cycloalkylalkyl, or carboxyalkyl, or R6 is formula IIa, IIb, or IIc; 
xe2x80x83or
xe2x80x94NR5R6 in xe2x80x94C(O)xe2x80x94NR5R6 for A represents a 5 or 6 membered saturated ring, unsubstituted or substituted with carboxy, alkyloxycarbonyl, hydroxy, alkoxycarbonylalkoxy, phenylalkyl, or phenylalkoxycarbonyl;
R7 is hydrogen, alkyl, alkenyl, alkylthioalkyl, aryl, heteroaryl, carboxyalkyl, carboxy, alkoxycarbonylalkyl, arylalkyl, or heteroarylalkyl; or a compound of formula III 
R7A is hydrogen or alkyl
R8 is xe2x80x94OR9 or xe2x80x94NR10R11 , wherein
R9 is hydrogen, alkyl, arylalkyl; R10 is hydrogen or alkyl; and R11 is alkyl, aryl, heteroaryl, arylalkyl, or the group xe2x80x94NR10R11 represents a 5 or 6 membered ring unsubstituted or substituted with carboxy, alkyloxycarbonyl, hydroxy, alkoxycarbonylalkoxy, phenylalkyl, or phenylalkoxycarbonyl;
R12 is alkyl, aryl, or arylalkyl;
Y is xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(O2)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94;
m is 0, 1, or 2;
X is xe2x80x94SO2, xe2x80x94COxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94SO2NHxe2x80x94, or xe2x80x94C(O)NR13xe2x80x94 wherein R13 is hydrogen, alkyl, aryl, or carboxyalkyl;
and
dimeric forms, and/or pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof, preferably pharmaceutically acceptable esters, and/or pharmaceutically acceptable salts thereof, and most preferably pharmaceutically acceptable salts thereof
Particularly, the present invention refers to the compounds of formula (I) wherein R1 is hydrogen or alkylcarbonyl, preferably R1 is hydrogen or acetyl, and more preferably R1 is hydrogen.
Further, the present invention refers to the above compounds wherein R2 is alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, cycloalkyl, halogenalkyl, carboxyalkyl, aryl, arylalkyl, arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, aryloxyalkyl, arylalkenyl, or aryl(alkoxycarbonyl)alkyl, preferably wherein R2 is alkyl, aryl or arylalkyl, more preferably wherein R2 is alkyl, phenyl or naphthyl, and most preferabyl wherein R2 is naphthyl.
A further preferred embodiment of the present invention are the above compounds wherein R3 and R4 are independently alkyl, phenyl, phenylalkinyl, phenylalkyl, or phenylalkenyl, preferably R3 is alkyl, phenyl, phenylalkinyl, or R4 is phenylalkinyl, phenylalkyl, or phenylalkenyl.
Another preferred embodiment of the present invention refers to the above compounds of formula (I) wherein A is xe2x80x94C(O)xe2x80x94NR5R6.
In another preferred embodiment the invention refers to the above compounds wherein X preferably is xe2x80x94SO2xe2x80x94 or xe2x80x94C(O)(O)xe2x80x94.
In a preferred embodiment of the present invention m is 0 or 1, preferably m is 0.
In a further preferred embodiment of the present invention the compound of formula (I) may be characterized in that R5 is alkyl, cycloalkyl, cycloalkylalkyl, carboxyalkyl, or arylalkyl, preferably that R5 is alkyl, arylalkyl, or cycloalkyl.
Particularly, the invention refers to the above compounds, wherein xe2x80x94NR5R6 in xe2x80x94C(O)xe2x80x94NR5R6 for A represents a 5 or 6 membered ring, e.g. a piperidinyl or pyrrolidinyl ring, preferably piperidinyl, unsubstituted or substituted with carboxy, alkyloxycarbonyl, hydroxy, alkoxycarbonylalkoxy, phenylalkyl, or phenylalkoxycarbonyl, preferably alkoxycarbonyl or carboxy. Preferably xe2x80x94NR5R6 is piperidinyl or pyrrolidinyl, optionally substituted with alkoxycarbonyl or carboxy, more preferably xe2x80x94NR5R6 is piperidinyl.
In a preferred embodiment R6 is alkyl, alkylcarbonylalkyl, cyanoalkyl, hydroxyalkyl, hydroxyalkyl-(hydroxyalkyl), alkoxycarbonylalkyl, arylalkyl, arylcarbonylalkyl, arylaminocarbonylalkyl, aryl(alkyl)aminocarbonylalkyl, aminocarbonylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, cycloalkyl, cycloalkylalkyl, carboxyalkyl, or xe2x80x94NR5R6 in the group xe2x80x94C(O)xe2x80x94NR5R6represents a 5 or 6 membered ring as defined above, unsubstituted or substituted with carboxy, alkyloxycarbonyl, hydroxy, alkoxycarbonylalkoxy, phenylalkyl, or phenylalkoxycarbonyl, or R6 is a group of the formula 
wherein R7, R7a, R8 and R12 are as defined above. Preferably, R7 is hydrogen, alkyl, alkenyl, alkylthioalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carboxyalkyl, carboxy, alkoxycarbonylalkyl; R7A is hydrogen or alkyl, and R8 is OR9 or xe2x80x94NR10R11, wherein R9 is hydrogen, alkyl, arylalkyl; R10 is hydrogen or alkyl; and R11 is aryl, heteroaryl, arylalkyl, or the group xe2x80x94NR10R11 forms a 5 or 6 membered saturated ring as described above for xe2x80x94NR5R6; Y is preferably xe2x80x94Oxe2x80x94; xe2x80x94Oxe2x80x94S(O2)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, more preferably xe2x80x94Oxe2x80x94S(O2)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94, and R12 is alkyl, aryl or arylalkyl.
In a preferred embodiment the invention comprises the above compounds, wherein R6 is alkyl, alkylcarbonylalkyl, cyanoalkyl, hydroxyalkyl, hydroxyalkyl-(hydroxyalkyl), alkoxycarbonylalkyl, arylalkyl, arylcarbonylalkyl, arylaminocarbonylalkyl, aryl(alkyl)aminocarbonylalkyl, aminocarbonylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, carboxyalkyl, or heterocyclyl, preferably wherein R6 is alkyl, alkylcarbonylalkyl, arylalkyl, arylcarbonylalkyl, heteroarylalkyl, carboxyalkyl, and more preferably wherein R6 is alkyl, alkylcarbonylalkyl, benzyl, tetrazolylethyl, phenylcarbonylmethyl, or oxadiazolylmethyl.
The invention comprises also compounds, wherein R6 is a group of formula (IIa) or (IIb) 
wherein R7 is hydrogen, alkyl, alkenyl, alkylthioalkyl, aryl, heteroaryl, carboxyalkyl, carboxy, alkoxycarbonylalkyl, arylalkyl or heteroarylalkyl; or R7 is a group of the formula III 
wherein Y is xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(O2)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94Oxe2x80x94C(O)xe2x80x94NHxe2x80x94 and R12 is aryl, preferably phenyl and R8 is OR9 or xe2x80x94NR10R11, wherein R9 is hydrogen, alkyl, arylalkyl; R10 is hydrogen or alkyl; and R11 is aryl, heteroaryl, arylalkyl.
In addition, the invention refers to the above compounds, wherein R6 is a group of the formula 
wherein R7 and R8 are as defined above.
Preferably, R7 is hydrogen in the above compounds and R8 is xe2x80x94NR10OR11 and R10 and R11 are as defined above. In these compounds R10 preferably is hydrogen or methyl and R11 preferably is aryl, more preferably R11 is phenyl, unsubstituted or substituted with alkoxycarbonyl, carboxy, or hydroxyalkyl.
Other preferred embodiments of the present invention refer to compounds wherein A is xe2x80x94C(O)xe2x80x94R3 or xe2x80x94CH(OH)xe2x80x94R4. R3 and R4 are as defined above.
In a most preferred embodiment the present invention comprises compounds of the formula 
wherein R1, R2, A and X are as defined above.
Preferred embodiments of the present invention are the compounds exemplified in the examples. Especially, the invention comprises the following compounds
a) (2S,4S)-1-[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-piperidine-4-carboxylic acid ethyl ester;
b) (2S,4S)-1-[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-piperidine-4-carboxylic acid;
c) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid benzyl-methyl-amide;
d) (2S,4R)-2-[2-[[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-methyl-amino]-acetylamino]-benzoic acid methyl ester;
e) (2S,4R)-2-[2-[[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-methyl-amino]-acetylamino]-benzoic acid;
f (2S,4R)-4-[[[[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-methyl-amino]-acetyl]-methyl-amino]-benzoic acid;
g (2S,4R)-4-[[[[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-methyl-amino]-acetyl]-methyl-amino]-benzoic acid methyl ester;
h (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid [[(4-hydroxymethyl-phenyl)-methyl-carbamoyl]-methyl]-methyl-amide;
i) (2S,4R)-3-{Benzyl-[4-mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-amino }-propionic acid;
j) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid cyclopropyl-[2-(1H-tetrazol-5-yl)-ethyl]-amide;
k) (2S,4R)-1-[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidin-2-yl]-3-methyl-butan-1-one;
l) (2S,4R)-[[4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-(3-methyl-butyl)-amino]-acetic acid;
m) (2S,4R)-2-(Benzyl-methyl-carbamoyl)-4-mercapto-pyrrolidine-1-carboxylic acid isopropyl ester;
n) (2S,4R)-2-(Benzyl-methyl-carbamoyl)-4-mercapto-pyrrolidine-1-carboxylic acid pentyl ester;
o) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid benzyl-[2-(1H-tetrazol-5-yl)-ethyl]-amide;
p) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid hexyl-methyl-amide;
q) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid methyl-(2-oxo-2-phenyl-ethyl)-amide;
r) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid methyl-(4-methyl-2-oxo-pentyl)-amide;
s) (2S,4R)-4-Mercapto-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carboxylic acid (3-methyl-butyl)-[1,2,4] oxadiazol-3-ylmethyl-amide;
t) (2S,4R)-2-((S) or (R)-1-Hydroxy-3-phenyl-prop-2-ynyl)-4-mercapto-pyrrolidine-1-carboxylic acid butyl ester;
u) (2S,4R)-4-Mercapto-2-(3-phenyl-propionyl)-pyrrolidine-1-carboxylic acid butyl ester;
v) (2S,4S)-1-[4-acetylsufanyl-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-piperidine-4-carboxylic acid ethyl ester;
w) (2S,4R)-4-[[[[4-Acetylsulfanyl-1-(naphthalene-2-sulfonyl)-pyrrolidine-2-carbonyl]-amino]-acetyl]-methyl-amino]-benzoic acid methyl ester.
These compounds show IC50 values in the radioimmunoassay (E on ECE-inhibition, see below) of about 5 nM to 1000 nM.
A process for the preparation of a compound as defined above comprising the reaction of a compound of formula 1 (Scheme 3) 
wherein R2, X and m are as defined above and PG is a sulfur protecting group, e.g. S-trityl, S-para-methoxybenzyl or S-acetyl, with the amine HNR5R6, wherein R5 and R6 are as defined above to give a compound of the formula 2 (Scheme 3). 
The invention also refers to pharmaceutical compositions comprising a compound as defined above and a pharmaceutically acceptable excipient.
A further embodiment of the present invention refers to the use of compounds as defined above as active ingredients in the manufacture of medicaments comprising a compound as defined above for the prophylaxis and treatment of disorders which are caused by endothelin-converting enzyme (ECE) activity especially myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, graft rejection, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
Further the invention refers to the use of compounds as described above for the treatment or prophylaxis of diseases which are associated with myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
In addition the invention comprises compounds as described above for use as therapeutic active substances, in particular in context with diseases which are associated with zinc hydrolase activity such as myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection.
The invention also comprises a method for the therapeutic and/or prophylactic treatment of myocardial ischaemia, congestive heart failure, arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis, Buerger""s disease, Takayasu""s arthritis, diabetic complications, lung cancer, prostatic cancer, gastrointestinal disorders, endotoxic shock and septicaemia, and for wound healing and control of menstruation, glaucoma, diseases associated with cytostatic, ophthalmological, and cerebroprotective indications, and organ protection, which method comprises administering a compound as defined above to a human being or animal.
The invention also relates to the use of compounds as defined above for the inhibition of zinc hydrolase activity.
The invention also refers to the above compounds whenever manufactured by a process as described below.
Compounds of formula (I) can be prepared by methods known in the art or as described below. Unless indicated otherwise, the substituents R1, R2, R3, R4, R5, R6, R7,, R7a R8, R9, R10, R11, R12, A, m, X and Y are as described above. All starting materials are known or can be prepared by known methods.
The synthesis of the intermediates (acids) for the preparation of compounds of the formula (I) are depicted in Scheme 1. The starting material is commercial available or is synthesized from hydroxyproline by methods known in the art and described for example in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, M. Bodanszky and A. Bodanszky, Springer Verlag, Berlin, 1984.
Step a of Scheme 1 describes the persilylation of hydroxy- and amino groups, e.g. by reaction of compound 1 with hexamethyldisilazan at 140xc2x0 C. followed by reaction with R2SO2Cl in THF or conversion to all other R2X described later or di-t-butyldicarbonate, NaHCO3 in dioxane, H2O (BOC protection).
For inversion of the configuration (via mesylate) the resulting alcohol 2 is treated with MeSO3H, Ph3P, DIAD or DEAD in toluene (room temperature to 80xc2x0 C.) or (via bromide) with LiBr, DEAD, Ph3P in THF (4xc2x0 C. to room temperature) or (via chloride) with Ph3P, CCl4 in CH2Cl2 (3xc2x0 C. to room temperature). In the case of retention of the configuration (via mesylate or tosylate) alcohol 2 can be transformed to a compound of formula 3 by reaction with MeSO2Cl, pyridine, DMAP or TosCl, pyridine, DMAP in CH2Cl2 (0xc2x0 C. to room temperature).
For the introduction of a protected thiol moiety, compounds of formula 3 are treated with e.g. triphenylmethanethiol or 4-methoxybenzylmercaptane and K-Ot-Bu in DMF (for Br: 0xc2x0 C. to room temperature; for Cl: 0xc2x0 C.; for Mesylate: room temperature to 100xc2x0 C.) or with potassium thioacetate in DMF room temperature to 100xc2x0 C. (step c).
In the case of Xxe2x80x94R2=BOC and R=/t-butyl, BOC deprotection can be accomplished with TFA in CH2Cl2 at xe2x88x9220xc2x0 C. to room temperature to give an amine. For the introduction of a new R2, the amine may be reacted with R2OCOCl/pyridine in THF or with (a) R2OH/Cl3COCl/quinoline (formation of the chloroformate) followed by reaction with NaH, in case new R2X is a carbamate. In case R2X is a sulfonamide the amine may be treated with R2SO2Cl, (i-Pr)2EtN, optionally in the presence of catalytic DMAP or DMAP-poly. in CH2 Cl2 at room temperature or in case R2X is amide the amine may be reacted with with R2COOH, EDCI, DMAP.
Hydrolysis of ester 4 (PG=Acetyl, step d) can be achieved with aqueous lithium hydroxide in THF (0xc2x0 C. to RT) or sodium hydroxide in ethanol to give acid 5, in the case of t-butyl esters the saponification can be accomplished with TFA in CH2Cl2.
For the reaction of compound 5 to compound 6 (step e) the Arndt-Eistert reaction may be used. Therefore in the case m=1the following procedure is used: addition of (COCI)2, cat DMF in CH2Cl2 at 0xc2x0 C. to room temperature to give the corresponding acid chloride followed by reaction with trimethylsilyldiazomethane in THF, CH3CN at 0xc2x0 C. to room temperature to give the corresponding diazoethanone and rearrangement to the methyl ester with silver benzoate in MeOH, THF at xe2x88x9225xc2x0 C. to room temperature, followed by ester cleavage with aqueous lithium hydroxide in THF (0xc2x0 C. to RT) or sodium hydroxide in ethanol to give compound 6. For m=2 compounds of formula 5 can be transformed into the corresponding Weinreb amide (e.g. HClxe2x80xa2HNMeOMe, NMM, EDCI, HOBT) and can be converted to an aldehyde (LAH, xe2x88x9278 to xe2x88x9230xc2x0 C. in THF). The obtained compound can be converted by a Horner-Emmons reaction (e.g. (EtO)2P(xe2x95x90O)CH2COOEt, NaH in THF) followed by reduction of the double bond (e.g. Mg in MeOH) and saponification of the ester with either aqueous lithium hydroxide in THF (0xc2x0 C. to RT) or sodium hydroxide to give compound 6.
BOC replacement against other R2X may follow by BOC-cleavage and treatment of the amine with the reagents described above.
Starting from the S-acetyl protected ester 4, saponification of the ester and the thioester moiety with 0.1M LiOH in THF or 1M NaOH in THF gives the thiol, which is treated with iodine and triethylamine or iPr2NEt in CH2Cl2 to yield the disulfid-diacid 7 (step f, scheme 1). 
The side chain A can be readily assembled as depicted in Scheme 2a prior to the coupling to the acids which were prepared according to scheme 1. A suitably protected aminoacid is treated with TPTU, 4-Methylmorpholine in CH2Cl2 and the approprioate amine NHR10R11, or with 2-chloro-4,6-dimethoxy-1,3,5-triazine, 4-methyl-morpholine, amine NHR10R11 and 4-dimethylamino-pyridin in DMF. Alternatively, the amide 2 can be prepared by transferring the acid 1 into the corresponding chloride (e.g. oxalyl chloride in toluene) or into the corresponding mixed anhydride (e.g. isobutyl chloroformate and N-ethylmorpholine in DMF) followed by treatment with the desired amine NHR10R11 in DMF.
Deprotected amine 3 can be obtained (step b) in the case of BOC-protection by treatment with TFA in CH2Cl2 and in the case of Z-protection by hydrogenation (e.g. 10%Pd/C, H2 in MeOH in the presence of HCl or 10%Pd/C, H2 in MeOH/NEt3).
Further modifications of the side chain may be achieved prior to deprotection: An ester moiety at the aromatic ring (Ra) may be reduced to the hydroxymethyl group on treatment with lithium borhydride in THF (50xc2x0 C., 2 h, step c). This maybe protected as a TBDMS ether using TBDMSCl and imidazole in DMF, 12 h, 5xc2x0 C. to room temperature (step d) or transferred into an ether by treatment with sodium hydride, alkyl iodide in DMF (step e).
Other amines may be prepared using the route outlined in Scheme 2b: namely the alkylation of the amine 4 with the bromo derivative 5 gives the corresponding secondary amine 6 (step f). 
Compounds of the formula (I) can be prepared as depicted in Scheme 3 from the acid 1 and a corresponding amine NHR5R6. For the synthesis of thio protected amide 2 several methods may be employed: TPTU, NMM or iPr2NEt, HNR5R6 in CH2Cl2 or EDCI, HOBT, HNR5R6 in THF, transformation of the acid into the corresponding chloride (e.g. oxalyl chloride in DMF) or into the corresponding mixed anhydride (e.g. i-propylxe2x80x94or ethyl chloroformate in DMF) followed by treatment with HNR3R4 in DMF (step a).
Deprotection of the thio moiety (step b) may be achieved in the case PG is either p-methoxybenzyl or trityl using triethylsilane in TFA, or triisopropylsilane in TFA, for PG is acetyl using lithium hydroxide in THF/water or sodium alkoholates in THF.
In the case PG is the homodimer, disulfide cleavage with tri-n-butylphosphine and water in 2,2,2-tri-fluoroethanol or DTT, 2M aq. K2CO3 in MeOH, THF gives the thiol compound 3.
In the case of R2Xxe2x95x90BOC, the final R2X can be introduced by BOC deprotection (e.g. TFA, CH2Cl2 at 0xc2x0 C. to room temperature) to get the amine which can be further modified.
In case R2X is a carbamate this amine may be reacted with R2OCOCl, pyridine or (i-Pr)2EtN in THF or CH2Cl2 or by reaction with (a) R2OH, Cl3COCl, quinoline (formation of the chloroformate) followed by reaction with NaH. In case R2X is a sulfonamide the starting compounds may be reacted with R2SO2Cl, (i-Pr)2EtN, cat DMAP in Cl2CH2 at room temperature. In case R2X is urea the starting compounds may be reacted with isocyanate in EtOH at room temperature. In case R2X is an alkylated urea the starting compounds may be reacted with isocyanate in EtOH at room temperature followed by reaction with the corresponding alkylhalogenide, K-OtBu at 0xc2x0 C. to room temperature. In case R2X is an amide, the starting compounds may be reacted with R2COOH, EDCI, DMAP (with anhydride formation, and subsequent addition of the starting amine at-10 xc2x0 C. to room temperature or as alternative with R2COOH/EDCI/DMAP at room temperature. In case R2X is a sulfamide (for R13 is H) the amine may be reacted with sulfamoyl chlorides in dioxane in the presence of an excess of triethylamine. The sulfamoyl chlorides may be synthesized from R2NH2 and chlorosulfonic acid in CH2Cl2 at 0xc2x0 C. to room temperature followed by reaction with PCl5 in toluene at 75xc2x0 C. Alternatively the sulfamoyl chlorides can be synthesized in acetonitrile with R2NH2 and sulfuryl chloride at 0xc2x0 C. to 65xc2x0 C. In case R2X is an alkylated sulfamide (R13 is not H) the sulfamide R2SONH-may be reacted with NaH, alkyl halide in DMF at 0xc2x0 C. to room temperature.
In the cases in which R6 contains a cyano moiety treatment with triethyl silane in TFA at 0xc2x0 C. to room temperature may give reduction to the amide concomitant to the S-PMB-ether cleavage. Selective thio deprotection may be accomplished with triethyl silane in TFA at 0xc2x0 C. for 15 min. A further modification of the residue at R6 may be the transformation of the cyano moiety into a tetrazole by treatment with sodium azide and ammonium chloride in DMF at 70-120xc2x0 C. followed by deprotection as described above (e.g. TFA, Et3SiH, reflux).
In the cases in which R6 is of the formula (II) with R8 is OR9, further modifications of the side chain can be achieved by ester saponification using lithium hydroxide in THF followed by amide formation with the amine HNR10R11 by one of the methods given before (step c,d, Scheme 3). The compound 5 can be deprotected according to the methods listed above (step e, see step b).
In the cases in which one of the residues R7 or R11 contains an ester this can be hydolyzed prior to S-deprotection (for S-PMB or Sxe2x80x94 Tr ) or for the homodimers using lithium hydroxide in THF or sodium hydroxide in THF.
A further method for the modification of the R11 comprises the reduction of an ester to a methylhydroxy group (e.g. with lithium borhydride in THF) or the deprotection of an alcohol moiety according to the methods known in the art (e.g. HF acetonitrile, CH2Cl2). Optionally this alkohol can be alkylated using e.g. sodium hydride, alkyl halogenides in THF or DMF or tert-Butyl 2,2,2-trichloroacetimidate with triflic acid in CH2Cl2, c-hexane or CCl4.
The hydroxy moiety of a tyrosine may be further manipulated by treating the compound 2 in case R12Y is a sulfonamide with R12SO2Cl, (i-Pr)2EtN, cat DMAP in CH2 Cl2 at room temperature, in case R12Y is a carbamate with R12OCOCl, pyridine or (i-Pr)2EtN in THF or CH2Cl2, in case R 12Y is urea the starting compounds may be reacted with isocyanate in EtOH at room temperature, in case R12Y is an amide, with R12COOH, EDCI, DMAP at-10xc2x0 C. to room temperature, in case R12Y is an ether with reactive alkyl halogenidesor arylalky halogenides. Deprotection of the thio moiety with trietylsilane, TFA for S-PMB and S-Tr or disulfide cleavage with tri-n-butylphosphine and water in 2,2,2-tri-fluoroethanol or DTT, 2M aq. K2CO3 in MeOH, THF gives compound 7 (steps f, b). Furthermore, compounds of the formula (I) with A is xe2x80x94CONR5R6 may be prepared as depicted in Scheme 4. The acid 1 can be treated with amine H2NR5 or H2NR6, EDCI, HOBT in THF or one of the other methods for amide formation described above followed by deprotection of the thio moiety with triethylsilane in TFA at reflux to give amide 3 (steps a, b). The second substituent may be introduced via alkylation. Therefore amide 2 may be treated with reactive R5-halogenides and sodium hydride in DMF to give after S-Tr or S-PMB deprotection with triethylsilane in TFA amide 4. In the case that R6 contains an ester, xcex1-alkylation leads to a modification of the side chain A (e.g. a. LiHMDS, b. R5xe2x80x94Br in THF). On deprotection the amide 5 can be obtained (steps d, b). 
Tetrazole derivatives may be prepared as depicted in Scheme 5. Acid 1 can be transformed into the amide 2 which contains a tetrazole moiety by treatment with an aminotetrazol, HOBT, EDCI in THF, followed by treatment with DBU in CH2Cl2 or using one of the methods described above (step b). For the synthesis of the tetrazolamino derivative see for example Sephane De Lombaert, Preparation of tetrazolylalkylaminomethylphosphonates as neutral endopeptidase inhibitors. U.S. Pat. No. 5,273,990 A 931228 or S. De Lombaert et al J. Med Chem, 2000, 43, 488-504 and literature sited therein. The tetrazole moiety may be protected before introduction of the side chain (e.g. PG2 is CH2CH2CN) or by manipulations of the amide 2 (e.g. 4-methoxybenzylchloride, NaI, Et3N in acetone).
Alkylation of the amide 2 which contains a PG2-protected tetrazole (e.g. sodium hydride, alkyl halogenide in DMF) gives after deprotection of the thio and the tetrazole moieties (e.g. triethylsilane, TFA, 80xc2x0 C.) the compound 3 (steps c,b).
In the case of the unprotected tetrazole derivative 2 (PG2 is H) alkylation using sodium hydride, alkyl halogenide in DMF may give a mixture of double alkylated regioisomers which can be separated and deprotected (e.g. triethylsilane, TFA, 80xc2x0 C.) to give compound 5 and 6 (step d,b).
Another method for the modification at the residue R2X is depicted in Scheme 6.
Treatment of the methylsulfonamide derivative 1 with LDA followed by alkylation with e.g. benzylbromide followed by transformation to the amide 2 by treatment with NHR5R6, HOBT, EDCI in THF or any other method described above for the amide formation (steps a,b). Dialkylation can be accomplished with LiHMDS, followed by treatment with e.g. benzylbromide followed by transformation to the amide 4. In both cases thiol deprotection may be achieved with triethysilane in TFA.
An alternative synthetic route is depicted in Scheme 7, in which the amide formation is performed prior to the introduction of the thio moiety. Acid 1 is preactivated with TPTU in CH2Cl2, NMM or by treatment with EDCI, CH2Cl2, NMM in CH2Cl2 followed by treatment with the amine NHR3R4 or by any other amide formation described in the previous schemes.
For inversion of the configuration (via mesylate) the resulting alcohol 2 is treated with MeSO3H,/Ph3P, DIAD or DEAD in toluene (room temperature to 80xc2x0 C.) or (via bromide) with LiBr, DEAD, Ph3P in THF (4xc2x0 C. to room temperature) or (via chloride) with Ph3P, CCl4 in CH2Cl2 (3xc2x0 C. to room temperature). In case of retention of the configuration (via mesylate or tosylate) alcohol 2 can be transformed to a compound of formula 3 by reaction with MeSO2Cl, pyridine, DMAP or TosCl, pyridine, DMAP in CH2Cl2 (0xc2x0 C. to room temperature).
Transformation to the corresponding protected thio compound 4 can be achieved e.g. by treatment with potassium thioacetate in DMF.
If further modification of the R2X moiety is desired, in the case of R2X is BOC deprotection can be achieved with TFA in CH2Cl2 followed by further modification of the liberated amine by treatment with the reagents described for reactions in Scheme 1 and 3.
Cleavage of the thioester 4 can be achieved by treatment with 0.1M lithium hydroxide in THF or 1M sodium hydroxide in THF (cleavage of all other ester moieties) or with sodium alkanolate in THF to give compound 5. 
Ketones and alcohols of formula (I, A=xe2x80x94C(O)xe2x80x94R3 or xe2x80x94CH(OH)xe2x80x94R4) may be prepared as depicted in Scheme 8. In the scheme the group R3 also includes the definitions for R4 as mentioned above. The starting acid 1 may be transferred into the Weinreb derivative with NHMeOMexc2x7HCl, Me3Al (excess) in toluene or by pre-activation of acid 1 with TPTU, Huenig""s base in DMF at RT followed by reaction with NH(OMe)Me.
In the case of R2X=BOC, the final R2X can be introduced by BOC deprotection (e.g. TFA, CH2Cl2 at 0xc2x0 C. to room temperature) to get the amine which can be further reacted with one of the reagents described for the compounds in Scheme 1 or 3.
The Weinreb derivative 2 may be treated with metal organic compounds (e.g. R3MgBr in THF or R3Li xe2x88x9225xc2x0 C. to RT in THF) to give the S-protected ketones 3. Deprotection may be accomplished by oxidation to the corresponding disulfide employing DMSO, Me3SiCl in acetonitril followed by disulfid cleavage with DTT in the presence of potassium carbonate in methanol or with triethylsilane in TFA to give ketone 4. Deprotection and reduction to the alcohols 5 may be achieved using iPr3SiH, TFA in CH2Cl2.
For the synthesis of ketones of the type 12 (steps i, b, c), the acid 1 can be transferred into the corresponding Weinreb derivative 10 by pre-activation with TPTU, Huenig""s base in DMF at RT followed by reaction with NHR5CH2NH(OMe)Me. From Weinreb derivative 10 the desired ketones can be prepared by treatment with metal organic compounds (e.g. R3MgBr in THF or R3Li xe2x88x9225xc2x0 C. to RT in THF) followed by deprotection using triethylsilane in TFA.
Alternatively, the alcohols 5 (for m=0) can be prepared from the ester 6 by selctive reduction to the aldehyde 7 (e.g. di-isobutylaluminium hydride in toluene, THF at xe2x88x9278xc2x0 C. (step e)) followed by treatment with metal organic compounds (e.g. R3MgBr in THF or R3Li xe2x88x9225xc2x0 C. to RT in THF, step f) to give alcohol 8 as a mixture of diastereomers. In the case R3 contains a triple bond, this can optionally be reduced (H2/Pd/C in MeOH at 1 atm). For the introduction of the protected thio moiety, compound 8 may be treated with potassium acetate in DMF at 100xc2x0 C., 2.5 h. After separation of the diastereomers the single compounds are liberated from the S-Ac prodrugs by treatment with 1N LiOH in EtOH (step h).
For the preparation of compounds of formula (I) the reaction pathway of Scheme 9,10-resin synthesis can be followed: the synthesis of the starting material 1 from hydroxyproline is described in Scheme 1. TFA, triisopropyl deprotection at reflux for 30 minutes gives thiol 2 (step a). The final R2X may be introduced either before attachment to the resin or after the preparation of the ketone on the resin (Scheme 10). For the second case, R2X ideally comprises FMOC. This may be prepared from R2X (xe2x95x90BOC) of starting acid 1 by methods known in the art and described for example in xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d, M. Bodanszky and A. Bodanszky, Springer Verlag, Berlin, 1984 to a nonacid labile protecting group (e.g. R2X=FMOC, first selective BOC-deprotection with 40% TFA in CH2Cl2 at RT followed by reaction with Fmocxe2x80x94OSu in dioxane/water and NaHCO3 as base).
The resin may be prepared as follows (step b, Scheme 9): The linker 4-(xcex1,xcex1-diphenylhydroxymethyl)benzoic acid is activated using TPTU, diisopropylethylamine in DMF and added to benzhydrylamine resin 3. The resin 4 is then treated with thiol 2 in CH2Cl2, TFA to give the resin loaded starting material 5.
The synthesis of final compounds of formula (I) is shown in Scheme 10: The resin linked acid 1 is transformed into the corresponding Weinreb derivative 2 by pre-activation of acid 1 (TPTU, Huenig""s base in DMF at RT) followed by reaction with NH(OMe)Me. The compound 2 is treated with metal organic compounds of type R5MX to give the resin bound ketones 3 which can be detached by treatment with TFA, iPr3SiH in CH2Cl2 at RT (step d).
Further modification of R2 can be accomplished in the case of R2X=FMOC before detaching the compound of the resin. Therefore, the FMOC protected compound 2 is treated with 20% piperidine in DMF, followed by introduction of the new R2X by the methods described for synthesis in Schemes 1 and 3. 
Preparation of prodrugs (Scheme 11) may be accomplished by treating the thiols 1 with the corresponding anhydrides or acid chlorides (e.g. Ac2O/pyridine in CH2Cl2 at room temperature or AcCl/ pyridine in CH2Cl2(step a)). If the residue A contains a carboxylic acid moiety this can be transferred into an ester (e.g. alcohol, EDCI, DMAP, CH2Cl2, step b).
Disulfide derivatives may be prepared by treating the thiol 1 with Ac-Cys(NPys)-OH in DMF as depicted in step c.
On the basis of their capability of inhibiting metalloprotease activity, especially zinc hydrolase activity, the compounds of formula I can be used as medicaments for the treatment and prophylaxis of disorders which are associated with vasoconstriction of increasing occurrences. Examples of such disorders are high blood pressure, coronary disorders, cardiac insufficiency, renal and myocardial ischaemia, renal insufficiency, dialysis, cerebral ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage, Raynaud syndrome and pulmonary high pressure. They can also be used in atherosclerosis, the prevention of restenosis after balloon-induced vascular dilation, inflammations, gastric and duodenal ulcers, ulcus cruris, gram-negative sepsis, shock, glomerulonephtritis, renal colic, glaucoma, asthma, in the therapy and prophylaxis of diabetic complications and complications in the administration of cyclosporin, as well as other disorders associated with endothelin activities.
The ability of the compounds of formula (I) to inhibit metalloprotease activity, particularly zinc hydrolase activity, may be demonstrated by a variety of in vitro and in vivo assays known to those of ordinary skill in the art.
A) Cell Culture
A stable human umbilical vein endothelial cell line (ECV304) was cultured in xe2x80x9ccell factoriesxe2x80x9d as described until confluency (Schweizer et al. 1997, Biochem. J. 328: 871-878). At confluency cells were detached with a trypsin/EDTA solution and collected by low speed centrifugation. The cell pellet was washed once with phosphate buffered saline pH 7.0 and stored at xe2x88x9280xc2x0 C. until use.
B) Solubilization of ECE from ECV304 cells
All procedures were performed at 0-4xc2x0 C. if not stated otherwise. The cell pellet of 1xc3x97109 cells was suspended in 50 ml of buffer A (20 mM Tris/HCl, pH 7.5 containing 5 mM MgCl2, 100 xcexcM PMSF, 20 xcexcM E64, 20 xcexcM leupeptin) and sonicated. The resulting cell homogenate was centrifuged at 100,000 gav for 60 minutes. The supernatant was discarded and the resulting membrane pellet was homogenized in 50 ml buffer A and centrifugated as described. The washing of the membrane fraction in buffer A was repeated twice. The final membrane preparation was homogenized in 50 ml of buffer B (buffer A+0.5% Tween 20 (v/v), 0.5% CHAPS (w/v), 0.5% Digitonin (w/v)) and stirred at 4xc2x0 C. for 2 hours. Thereafter the remaining membrane fragments were sedimented as described. The resulting clear supernatant containing the solubilized ECE was stored in 1.0 ml aliquots at xe2x88x92120xc2x0 C. until use.
C) ECE Assay
The assay measured the production of ET-1 from human big ET-1. To measure high numbers of samples an assay performed in 96 well plates was invented. The enzyme reaction and the radioimmunological detection of the produced ET-1 was performed in the same well, using a specifically developed and optimized coating technique.
D) Coating of Plates
Fluoronunc Maxisorp White (code 437796) 96 well plates were irradiated with 1 joule for 30 minutes in a UV Stratalinker 2400 (Stratagene). The 96 well plates were then fill with 300 xcexcl protein A solution (2 xcexcg/ml in 0.1 M Na2CO3 pH 9.5) per well and incubated for 48 hours at 4xc2x0 C. Coated plates can be stored for up to 3 weeks at 4xc2x0 C. until use.
Before use the protein A solution is discarded and the plates are blocked for 2 hours at 4xc2x0 C. with 0.5% BSA in 0.1M Na2CO3, pH 9.5.
Plates were washed with bidestilled water and were ready to perform the ECE assay.
E) Screening Assay
Test compounds are solved and diluted in DMSO. 10 xcexcl of DMSO was placed in the wells, followed by 125 xcexcl of assay buffer (50 mM Tris/HCl, pH 7.0, 1 xcexcM Thiorphan, 0,1% NaN3, 0.1% BSA) containing 200 ng big ET-1. The enzyme reaction was started by the addition of 50 xcexcl of solubilized ECE (diluted in assay buffer 1:30 to 1:60 fold (v/v)). The enzyme reaction was carried out for 30 minutes at 37xc2x0 C. The enzyme reaction was stopped by addition of 10 xcexcl 150 mM ETDA, pH 7.0.
Radioimmunoassay:
The ET-1 RIA was performed principally as described earlier (Lxc3x6ffler, B. -M. and Maire, J. -P. 1994, Endothelium 1: 273-286). To plates containing the EDTA stopped enzyme reaction mixture 25 xcexcl of assay buffer containing 20000 cpm (3-(125I)Tyr)-endothelin-1 and 25 xcexcl of the ET specific antiserum AS-3 (dilution in assay buffer 1:1000) was added. Plates were incubated under mixing at 4xc2x0 C. over night. Thereafter, the liquid phase was sucked with a plate washer and plates were washed once with bidestilled water. To the washed plates 200 xcexcl scintillation cocktail (Microscint 40 LSC-Cocktail, Packard, code 6013641) was added and plates were counted for 2 minutes per well in a Topcount.
Standard curves were prepared in plates with synthetic ET-1 with final concentrations of 0 to 3000 pg ET-1 per well. In all plates controls for maximal ECE activity (in the presence of 10 xcexcl DMSO) and for background production of ET-1 immunoreactivity (in the presence of 10 mM EDTA or 100 xcexcM phosphoramidon) were performed. Assays were run in triplicate.
F) Kinetic Assay
The described assay format could be used to determine the kinetic characteristics of the used ECE preparation as well as different ECE inhibitors (i.e. Km, Ki) by variation of the substrate concentration used in the assay.
G) Cell based ECE Assay
Human ECE-1c was stable expressed in MDCK cells as described (Schweizer et al. 1997, Biochem. J. 328: 871-878). Cells were cultured in 24 well plates to confluency in Dulbecco""s modified Eagles""s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 0.8 mg/ml geneticin, 100 i.u./ml penicillin and 100 xcexcg/ml streptomycin in a humidified air/CO2 (19:1) atmosphere. Before ECE assay the medium was replaced by 0.5 ml DMEM-HBSS 1:1, 10 mM HEPES pH 7.0 supplemented with 0.1% (w/v) BSA. The inhibitors were added in DMSO at a final concentration of 1%. The enzyme reaction was started by the addition of 0.42 xcexcM human big ET-1 and performed for 1.5 hours at 37xc2x0 C. in an incubator. At the end of incubation, the incubation medium was quickly removed and aliquots were analysed by radioimmunoassay for produced ET-1 as described above.
The ECE screening assay was validated by the measurement of the characteristic inhibitor constants of phosphoramidon (IC50 0.8xc2x10.2 xcexcM) and CGS 314447 (IC50 20xc2x14 nM) [De Lombaert, Stephane; Stamford, Lisa B.; Blanchard, Louis; Tan, Jenny; Hoyer, Denton; Diefenbacher, Clive G.; Wei, Dongchu; Wallace, Eli M.; Moskal, Michael A.; et al. Potent non-peptidic dual inhibitors of endothelin-converting enzyme and neutral endopeptidase 24.11. Bioorg. Med. Chem. Lett. (1997), 7(8), 1059-1064]. The two inhibitors were measured with IC50 values not significantly different from those described in the literature but measured with different assay protocols. In the cell based assay phosphoramidon showed an IC50 of 4 xcexcM. This assay gave additional information about the inhibitory potency of inhibitors under much more physiologic conditions, as e.g. the ECE was embedded in a normal plasma membrane environment. It is important to state, that the screening assay was performed in the presence of 1 xcexcM Thiorphan to block any potential big ET-1 degradation due to the action of NEP24.11. No NEP activity was present in MDCK-ECE-1 c transfected cells in preliminary experiments when ET-1 production was measured in presence or absence of thiorphan. In subsequent experiments no thiorphan was added in the incubation medium.
According to the above methods, the compounds of the present invention show IC50 values in the radioimmunoassay (E on ECE-inhibition) of about 5 nM to about 1000 xcexcM. The preferred compounds show values of 5 nM to 1000 nM.
As mentioned earlier, medicaments containing a compound of formula I are also an object of the present invention as is a process for the manufacture of such medicaments, which process comprises bringing one or more compounds of formula I and, if desired, one or more other therapeutically valuable substances into a galenical administration form.
The pharmaceutical compositions may be administered orally, for example in the form of tablets, coated tablets, dragxc3xa9es, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally or percutaneously, for example using ointments, creams, gels or solutions; or parenterally, for example using injectable solutions.
For the preparation of tablets, coated tablets, dragxc3xa9es or hard gelatin capsules the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients. Examples of suitable excipients for tablets, dragxc3xa9es or hard gelatin capsules include lactose, maize starch or derivatives thereof, talc or stearic acid or salts thereof.
Suitable excipients for use with soft gelatin capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc.; according to the nature of the active ingredients it may however be the case that no excipient is needed at all for soft gelatin capsules.
For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose.
For injectable solutions, excipients which may be used include for example water, alcohols, polyols, glycerin, and vegetable oils.
For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
The pharmaceutical compositions may also contain preserving agents antioxidants, solubilising agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable agents.
The dosages in which the compounds of formula I are administered in effective amounts depend on the nature of the specific active ingredient, the age and the requirements of the patient and the mode of application. In general, dosages of 0.1-100 mg/kg body weight per day come into consideration, although the upper limit quoted can be exceeded when this is shown to be indicated.
The following specific examples are provided as a guide to assist in the practice of the invention, and are not intended as a limitation on the scope of the invention.